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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2016 to 17 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Apperance: Viscous gold coloured liquid
Storage conditions: At ambient temperature, protected from light in a sealed container

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST was used because of the historical control data available.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A correction factor (including purity) of 1.429 was used when calculating quantities of test item to be used during dose preparation.
Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Vehicle was added to the mixture (to achieve approximately 50 % of the final volume) and stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (nominally 2-8 °C).

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: 4.57, 11.43 and 28.58 mg/mL
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined.
Homogeneity and stability of the test item in the vehicle at concentrations of 2 mg/mL and 200 mg/mL (2.858 mg/mL and 285.8 mg/mL as supplied) was achieved following re-suspension of the formulations (by 20-fold inversion and magnetic stirring for a minimum period of 20 minutes) following refrigerated storage (2-8 ºC) for up to 19 days. Formulations were also stable at ambient temperature (nominally 21 °C) for up to 48 hours.
Achieved concentration Samples of each formulation prepared for administration for the first formulation occasion (Week 1), during Weeks 5 and 6 and the last formulation occasion (Week 7) were analysed for achieved concentration of the test item.

ANALYTICAL PROCEDURE
The analytical method involved extraction and dilution in propan-2-ol followed by liquid chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to single matrix matched bracketing standards at 500 ng/mL. Calibration standards and samples were matrix matched. Procedural recovery samples were prepared concurrently with samples and results were corrected for the mean recovery value at each level at analysis.

CONCENTRATION OF DOSE FORMULATIONS
The formulations for the First Formulation occasion, Week 5, Week 6 and the Last Formulation occasion were sampled (4 x 1 mL, accurately weighed) by Pharmacy personnel. Duplicate samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. On the First Formulation occasion and for Week 5, contingency samples for Groups 2, 3 and 4 were analysed due to problems with the initial analysis. Samples were disposed of once analysis was complete.

RESULTS AND CONCLUSION
For the Week 1 samples, the procedural recovery results were originally outside acceptance criteria. This was investigated by re-diluting recovery and formulation samples in duplicate. Because the results from this investigation would not be available until the contingency samples had passed their valid stability period it was decided to analyse the contingency samples as a precautionary measure.
For the Week 5 samples, the original analysis could not be reported due to analytical errors which meant the recovery samples were outside the acceptance criteria and in some cases were as high as 200 %. Contingency samples were therefore analysed for this occasion. The mean concentrations were within applied limits +10 %/-15 % of the nominal concentration, confirming the accuracy of formulation.
The coefficients of variation for Groups 2 and 4 on the First Formulation occasion were >5 %, this is considered to be due to the variability of the LC-MS/MS method and formulations were deemed to have been prepared correctly. On all other occasions, the differences from the mean of individual results were <±5 % indicating precise analysis.
Procedural recovery results throughout the study were variable on a run by run basis as a result of the inherent variability of the LC-MS/MS method used for analysis. After a review of the completed study data, it is considered that an acceptance limit of 90 – 110 % of the nominal fortification is acceptable for recoveries. This has been applied to all analytical occasions and was reviewed for each run.
Mean analysed concentrations were calculated using unrounded figures. Analysed concentrations are corrected for the mean recovery values at each level.
Details on mating procedure:
MATING PROCEDURE
- M/F ratio per cage: 1:1 from within the same treatment group.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear; checked daily.
- After successful mating each pregnant female was caged: Individually during gestation.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Duration of treatment / exposure:
- Males: Daily for a minimum of four weeks.
- Females: Daily for 15 days before pairing and until Day 6 of lactation.
Animals of the F1 generation were not dosed.
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
4 to 6 weeks (at least 4 weeks)
Doses / concentrationsopen allclose all
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a two-week preliminary toxicity study and a four-week toxicity study conducted on the test material.
In the two-week preliminary toxicity study, doses of 300 and 1000 mg/kg/day were not tolerated resulting in the premature termination of both sexes receiving 1000 mg/kg/day and females receiving 300 mg/kg/day after a few days. A dose of 100 mg/kg/day was well tolerated in both sexes and elicited no clear test substance-related signs, with macroscopic gastrointestinal findings confined to a single female with a thickened non-glandular region of the stomach. In the 4-week repeat dose toxicity study, doses of 16, 40 and 100 mg/kg/day were well tolerated and did not result in any mortality or signs of other adverse systemic toxicity based on the study investigations performed up to the end of the 4-week treatment period and excluding results of the histopathological assessment.
Based on this information, it was considered appropriate to investigate a high dose level of 100 mg/kg/day in this current study. The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. The groups were adjusted to reduce inter- /intra-group variation.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacements were made before allocation of two males due to body weight range extremes.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs associated with dosing were checked daily during the first week of treatment, weekly thereafter and for females on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose.
- After completion of dosing the group.
- One to two hours after dosing.
- As late as possible in the working day.
A detailed physical examination was performed weekly for males and for females, weekly before pairing, on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes
The weight of animals was recorded as follows:
F0 females During acclimatization.
- Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
- Days 0, 3, 7, 10, 14, 17 and 20 after mating.
- Day 1, 4, and 7 of lactation.
- On the day of necropsy.
Group mean values and standard deviation (SD) were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (and SD) was calculated separately for males and females; the group mean values were derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals.
Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males
- Weekly before pairing (from first day of treatment until pairing).
F0 females
- Weekly before pairing (from first day of treatment until pairing).
- Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating.
- Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
Group mean food consumptions and standard deviations were derived from unrounded cage values.

OTHER:
VAGINAL SMEARS
Wet smears were taken daily after pairing until mating using pipette lavage.

PARTURITION OBSERVATIONS AND GESTATION LENGTH
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

PRE-COITAL INTERVAL
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals of durations of 1-4, 5-8, 9-12 and 13-14 days of pairing was calculated.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes. For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
Statistics:
See below.
Indices:
MATING PERFORMANCE AND FERTILITY
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals pairing) x 100


GESTATION LENGTH AND INDEX
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = Number of live litters born Number pregnant x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs observed were minor and not attributed to treatment.
There were no treatment related changes observed in association with the time of dosing. Salivation was observed in males only towards the end of their respective treatment period (Day 25) at an increasing incidence with increasing dose. However, this only appeared on a single day, and the sign itself is most likely to reflect a general distaste of the formulation, therefore it is not considered a toxic effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gain for males throughout their 4 week treatment period, and for females before pairing, and throughout gestation and lactation, was similar to Controls and unaffected by treatment at all dose levels investigated.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect on food consumption before pairing in males or females, or during gestation and lactation for females, at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Mean testis and epididymis weights were slightly lower than Controls at 100 mg/kg/day although no statistical significance was attained. Comparing the current data to historical control data for testis and epididymis weights (mean 3.493, range 3.380-3.652 for testis and mean 1.162, range 1.116-1.198 for epididymis) these changes were considered to represent normal biological variation for animals of this strain and ages at this facility and therefore these changes are considered not to be related to treatment. Seminal vesicle weights were slightly lower than Controls at 40 or 100 mg/kg/day, without dose-relationship, and with only values adjusted for terminal body weight attaining statistical significance at both dose levels.
There were no effects of treatment upon male pituitary and prostate weights, or female ovary weights, at any of the dose levels investigated.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings in adult males or females at necropsy that would infer any treatment related changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings in adult males or females at necropsy that would infer any treatment related changes.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Pre-coital interval was unaffected by treatment, with only one pairing on study (in the Control group) which did not mate within the first 4 days of pairing.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no effect on the number of live births.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect on post-implantation survival.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no effect on the number of live births.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no clear effect of treatment upon mean gestation length or the gestation index.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There was no clear effect of treatment upon mean gestation length or the gestation index.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Treatment had no effect upon mating performance or fertility.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment-related effects.

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean body weight of offspring on Day 1 of age, and subsequent body weight gain during Days 1-7 of age was similar to Controls across all treatment groups and considered unaffected by treatment at all dose levels investigated.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Mean litter size was similar to controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of test material on sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter size was similar to controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
OFFSPRING CLINICAL SIGNS
There was no change in the clinical condition of offspring that was considered related to maternal treatment.

OFFSPRING MACROPATHOLOGY
Necropsy findings observed in offspring dying prematurely or in those killed at scheduled termination revealed no findings that were considered to relate to parental treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatement related effects

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation Analysis Results

The mean concentrations were within applied limits +10 %/-15 % of the nominal concentration, confirming the accuracy of formulation. The coefficients of variation for Groups 2 and 4 on the First Formulation occasion were >5 %. This was considered to be due to the variability of the LC-MS/MS method and formulations were deemed to have been prepared correctly. On all other occasions, the differences from the mean of individual results were±<5 % indicating precise analysis.

Discussion

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 16, 40 or 100 mg/kg/day was well tolerated with no adverse effect of treatment. There was a marginal reduction in testis and epididymis weights in males which received 100 mg/kg/day. Compared to historical control data, these changes were considered to represent normal biological variation for animals of this strain and ages at this facility. Slightly lower seminal vesicle weights amongst males receiving 40 or 100 mg/kg/day were also recorded however, no differences in organ weights were apparent at the same doses on the four-week toxicity study. In addition, there was no microscopic correlate from evaluation of the testes, epididymides and seminal vesicles from males which received 40 or 100 mg/kg/day either on the four-week toxicity study, or following evaluation of the testes and epididymides on the current study, and fertility and fecundity (litter size) were unaffected at all dose levels investigated in the current study. Therefore these differences in organ weights were considered not to represent an adverse effect of treatment and likely to be unrelated to treatment.

Treatment had no effect upon mating performance or fertility and there was no clear effect of treatment upon mean gestation length or the gestation index.

The number of offspring born, subsequent survival and growth to Day 7 of age and clinical condition of the offspring were unaffected by treatment at dose levels up to 100 mg/kg/day. There were no macroscopic findings in offspring that were considered to relate to parental treatment.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, oral administration of the test material to male and female Han Wistar rats was well tolerated, with no effect of treatment upon reproductive or developmental parameters observed up to the highest dose level tested of 100 mg/kg/day.
Executive summary:

The potential for the test material to cause reproductive/developmental effects, was investigated in a GLP study conducted in accordance to the standardised guideline OECD 421.

Three groups of ten male and ten female rats received the test material at doses of 16, 40 or 100 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance and fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken.

The clinical condition, litter size, survival, sex ratio, body weight and macropathology for all offspring were also assessed.

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 16, 40 or 100 mg/kg/day was well tolerated, with no deaths and no effect of treatment upon the clinical condition of animals.

There was no effect of treatment on body weight gain or food consumption at any of the dose levels investigated and pre-coital interval, mating performance and fertility and gestation length were unaffected.

The number of offspring born, offspring sex ratio and offspring survival and growth to Day 7 of age were unaffected at dose levels up to 100 mg/kg/day and there were no clinical observations or macroscopic necropsy findings amongst offspring that were considered to relate to parental treatment.

There were no treatment related macroscopic or microscopic findings, and no differences in organ weights, which were considered to be related to treatment.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) for at least 4 weeks at dose levels of 16, 40 or 100 mg/kg/day was well tolerated, with no effect of treatment upon reproductive or developmental parameters observed up to the highest dose level tested of 100 mg/kg/day. The NOAEL is therefore > 100 mg/kg bw/day.